Low-cost, high-throughput gene synthesis and precise control of protein expression are the critical technical problems of synthetic biology. Currently, the main method for artificially synthesizing gene is to obtain long chain DNA by splicing and assembling short chain oligonucleotides. The cost of the method for artificially synthesizing short chain oligonucleotide by chemical method is very high (RMB 0.6 per nucleotide), and the error rate of synthesis remains as high as one base deletion per 100 bases and one mismatch or insertion per 400 bases. As a result, synthesis of gene or genome from assembling oligonucleotide is expensive, and the accumulative error rate of synthesis is high. Repairing errors by clone sequencing and mutagenesis methods further increases the amount of labor and total cost.
Oligonucleotide synthesis by performing large scale parallel synthesis on microfluidic chip can significantly reduce the cost. Currently, the method for synthesizing oligonucleotide on the chip mainly includes ink-jet printing (Agilent), 5′-end modified photolabile protecting groups (Nimblegen/Affymetrix), photo-generated acid deprotection (Atactic/Xeotron) and electrochemical method (Oxamer/Combimatrix). However, due to the very small surface area of the microfluidic chip, the yield of the oligonucleotide synthesis is low, with the concentration of each sequence of oligonucleotide in the solution being 10−12 M or less. Thus, extensive amplification is required prior to assembling into gene. Currently, a feasible method is as follows: releasing the synthesized oligonucleotide from the microfluidic chip by chemical or enzymatic treatment, amplifying by PCR, digesting by restriction enzyme, and purifying, then assembling the obtained oligonucleotide into gene or genome, and the error repair method of the gene synthesis mainly involves polyacrylamide gel electrophoresis and HPLC. Since the current methods of gene synthesis and error repair are still compromised by cumbersome steps, new strategies are urgently required to increase integration and miniaturization, lower cost, and increase the efficiency in synthesis and error repair.